The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Traditionally, radioimmunoassays were in the beginning performed in solution using test-tubes and complicated separation methods. With the advent of solid phase technology radioactive isotopes have been replaced by diverse labels, and especially monoclonal antibodies have been used in an increasing number of applications.
The aforementioned improvements have made a wider range of traditional immunoassays and other bioaffinity assay methods available (e.g. Alternative Immunoassays, W. P. Collins, Wiley, Chichester 1985, and Luminescence Immunoassay and Molecular Applications, Ed. Knox van Dyke, CRC Press, Boston 1990, and R. P. Ekins et al., Clin. Chem. 1991; 37:1955–1967). Solid phase assays have become routine assay methods both in the field of competitive and non-competitive assays, and the traditional test-tube has often been replaced by alternative solid phase facility. The microtiter plate was introduced as a solid phase for routine hormone assays in 1984 when Wallac Oy (Turku, Finland) introduced their DELFIA technology. Other commercial enterprises have also adopted the microtiter plate in similar routine assays, which, however, are based on different labelling technologies.
Microparticles manufactured of different materials have for some time been available as an alternative solid phase for bioaffinity assays (e.g. Molday W. J. et al., J. Cell. Biol.: 1975: 64, 75, and Nustad K. et al.: Microspheres. Medical and biological applications, Ed. A. Rembaum and Z. A. Tökes, Boca Raton, Fla., CRC Press 1988). Compared to microtiter plates the microparticles offer several advantages, for instance an immobilization method for bioaffinity reactants more easily adaptable to production scale, and a greater reaction velocity obtainable when using microparticles. The capacity of the microsphere-associated solid phase is also easily controllable by simply dispensing an amount of particles optimal for the method in question. The greatest disadvantages of microparticles have been traditionally associated with liquid handling (e.g. washes). With the introduction of magnetic microspheres this problem has been partly overcome, and in recent years an increasing number of commercial automated immunoassay methods have been based on their use. Nowadays novel manufacturing methods have allowed the production of microparticles of exact and reproducible size (e.g. of a diameter of 0.04–100 μm). Also the immobilization of the bioaffinity reactants onto the microparticles can be effected by various chemical methods.
The competitive biospecific assay method may be described as follows: The microparticles are coated with the bioaffinity reagent A, for whose binding sites, on the one hand, the analyte contained in the sample competes, and on the other hand, the bioaffinity reagent B, in this case an appropriately labelled analyte, also competes for the said binding sites. In the case of immunoassay, bioaffinity reagent B will be a labelled antigen, the antigen being the same as the antigen contained in the sample or to a suitable labelled derivative thereof. Because the analyte contained in the sample and the labelled analyte compete for the binding sites of the bioaffinity reactant A, the amount of label bound to the microparticle via reactant A, and consequently the signal from the microparticle, will be inversely related to the concentration of the analyte in the sample.
The non-competitive biospecific assay method may be described as follows: The microparticles are coated with the bioaffinity reactant A, whose binding sites only bind the analyte contained in the sample. The bioaffinity reactant B, which in this case is a suitably labelled reactant directed against the analyte contained in the sample, is bound by the analyte, which is linked to the microparticle via the bioaffinity reactant A. In the case of immunoassay, bioaffinity reactant B will be a labelled antibody directed against the antigen contained in the sample. The amount of label bound to the microparticle via reactant A, and consequently the signal strength from the microparticle, will be directly related to the concentration of the analyte in the sample.
The sensitivity of the competitive and non-competitive immunoassay has been extensively discussed earlier (Ekins R. P. et al., Pure and Appl. Chem., 1985; 57: 473–482, and Ekins R. P. et al., Clin. Chem., 1991; 37: 1955–1967). As regards the present invention, it is essential to consider the factors affecting the sensitivity and functionality of each assay principle.
Maximal sensitivity in a competitive immunoassay when using an antibody (a bioaffinity reactant) with an affinity constant K will be obtained by dividing by K the relative error of the signal strength at zero concentration. The error of the signal strength will be affected by two components, an experimental error component due to pipetting and other manipulations, and a signal strength measurement component, e.g. the statistical error of signal strength measurement. For the sake of simplicity, let us assume that the error due to signal strength measurement is 0, which is usually the case when the specific activity of the label is high. The maximal attainable sensitivity of the competitive assay is now ε/K where ε is the combined relative error due to experimental factors. Let us assume, for instance, that the experimental error is of the order of 1%; the maximal attainable sensitivity then, using an antibody with an affinity constant as high as possible, for instance,
1012l/mol, is of the order of 0.01 picomol/l. The sensitivity definition also demonstrates that unless the experimental error can be almost completely eliminated (<0.1%), there is no sensitivity advantage to be gained in using a label with extremely high specific activity.
Likewise, the factors affecting the sensitivity of the non-competitive are the following:
a) the relative error of the signal strength (α) when the analyte is omitted, i.e., the error due to non-specific binding of the labelled antibody (bioaffinity reactant)
b) the relative amount of non-specifically bound labelled antibody (k), and
c) the affinity constant (K) of the antibody
The sensitivity of the non-competitive assay is then k/K×α. The sensitivity of both competitive and non-competitive assay is inversely related to the affinity constant of the antibody used, but the non-competitive assay is affected by the relative error due to, on the one hand, non-specific binding of the labelled antibody and on the other hand, the relative binding of the labelled antibody, both of which could be assigned, for instance, a value of 1%. The maximal sensitivity of the non-competitive assay is then 0.0001×1/K or 0.1 fmol/l, if the affinity constant of the antibody used is 1012l/mol, which is usually considered to be the highest possible value. In a non-competitive assay it is advisable to consider the specific activity of the label, because it affects factors a) and b), and offers a possibility to improve the sensitivity of the assay.
In assays based on bioaffinity reactions factors structurally limiting the sensitivity of various determinations must also be considered; this is also essential as regards the present invention, because measurements will be taken from individual microparticles with extremely small amounts of analytes present.